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Introduction: The Angiotensin-Converting Enzyme 2 (ACE2) is the main receptor of the SARS-CoV-2. There is contradictory evidence on how the exposure to nicotine may module the concentration of soluble ACE2 (sACE2). The aim of this study was to assess the association between nicotine and sACE2 concentrations in saliva samples.Methods: Pooled analysis performed with data retrieved from two studies (n = 634 and n = 302). Geometric mean (GM) concentrations of sACE2, both total and relative to the total amount of protein in the sample, were compared according to sociodemographic variables and variables associated to nicotine. Multivariable linear regression models were fitted to explore the associations of sACE2 with nicotine adjusting for sex, age and body mass index. Spearman's rank-correlation coefficients were estimated between the concentrations of nicotine and cotinine, and pack-years, the concentration of relative sACE2 and the isoforms of sACE2.Results: We observed a significant increase of 0.108 & PTSTHOUSND; and 0.087 ng/mu l in the relative and absolute salivary sACE2 GM concentrations, respectively, between the lowest and highest nicotine levels. Similar results were observed for cotinine. These associations did not change in the multivariable linear models. There was a low correlation of nicotine and cotinine concentration with the concentration of relative salivary sACE2 (r(s) = 0.153 and r(s) = 0.132, respectively), pack-years (r(s) = 0.222 and rs = 0.235, respectively) and with the concentration of isoform 40 KDa (r(s) = 0.193 and r(s) = 0.140, respectively).Conclusion: Salivary nicotine concentration seems to be limitedly associated with the concentration of sACE2.

Cells maintain their size within limits over successive generations to maximize fitness and survival. Sizer, timer, and adder behaviors have been proposed as possible alternatives to coordinate growth and cell cycle progression. Regarding budding yeast cells, a sizer mechanism is thought to rule cell cycle entry at Start. However, while many proteins controlling the size of these cells have been identified, the mechanistic framework in which they participate to achieve cell size homeostasis is not understood. We show here that intertwined APC and SCF degradation machineries with specific adaptor proteins drive cyclic accumulation of the G

Atypical cyclins have recently emerged as a new subfamily of cyclins characterized by common structural features and interactor pattern. Interestingly, atypical cyclins are phylogenetically close to canonical cyclins, which have well-established roles in cell cycle regulation and cancer. Therefore, although the function of atypical cyclins is still poorly characterized, it seems likely that they are involved in cancer pathogenesis as well. Here, we coupled gene expression and prognostic significance analysis to bibliographic search in order to provide new insights into the role of atypical cyclins in cancer. The information gathered suggests that atypical cyclins intervene in critical processes to sustain cancer growth and have potential to become novel prognostic markers and drug targets in cancer.

Resumen Los clickers o Audience Response Systems (ARS) permiten a los alumnos responder preguntas planteadas por el profesor a través de dispositivos móviles y sus respuestas aparecen instantáneamente en la pantalla del aula, de manera que se pueden discutir y analizar en grupo facilitando en gran medida un aprendizaje socrático y dirigido por iguales (peer instruction). En la Facultad de Medicina de la UIC hemos empleado uno de los mencionados ARS, Socrative, para analizar el conocimiento previo, comprobar la adquisición autónoma de conocimiento, la adquisición de conocimientos después de una clase magistral y como repaso antes de la evaluación. Una encuesta anónima entre los estudiantes valora este tipo de sistemas en comparación con una metodología basada en la lectura de clase magistral. Los resultados no muestran diferencias estadísticamente significativas entre el método tradicional y el Socrative en cuanto al grado de interés ni a los conocimientos adquiridos. Sin embargo, las calificaciones obtenidas por los estudiantes con Socrative fueron significativamente superiores a las obtenidas mediante metodología tradicional. Además el 89% de los estudiantes opina que Socrative es fácil de usar, el 86% que mejora la participación en clase, el 85% que la ameniza, el 81% que facilita la autoevaluación; sin embargo, solo un 54% opina que Socrative le ayudó a obtener mejores resultados en la evaluación. Abstract The clickers or Audience Response Systems (ARS) allow students to answer questions posed by the teacher through mobile devices and their answers appear instantly on the classroom screen, so that they can be discussed and analysed as a group, greatly facilitating Socratic and peer-directed learning. In the Faculty of Medicine of the UIC, we have used one of the aforementioned ARS, — Socrative— to analyse the following aspects: prior knowledge, the autonomous acquisition of knowledge, the acquisition of knowledge after a master class and, finally, as a way to produce a review before the evaluation. We carried out an anonymous survey among our students to compare lectures where Socrative was used with classical lectures based on the professor delivery of the content. The results show no statistically significant differences between the traditional method and the Socrative in terms of the degree of interest or the acquired knowledge. However, the grades obtained by students with Socrative were significantly higher than those obtained when traditional methodology was used. In addition, 89% of students think that Socrative is easy to use, 86% that improves participation in class, 85% that enlivens it and 81% that facilitates self-evaluation; however, only 54% think that Socrative helped them to improve their marks.

For years it has been discussed the dichotomy between traditional in-person teaching versus online instruction. The lockdown, consequence of the Covid-19 pandemic, gave us an opportunity to gain evidence and compare in-person versus online teaching by the same professor, in the same subject and by the same students which is an interesting scenario because the only variable is the teaching setup. Here, we conducted a survey of students that following a traditional in-person subject were abruptly compelled to continue it online. We summarize the students’ responses when asked to compare both types of teaching with the singularity that the rest of variables were kept constant. Finally, we compared the grades of this “hybrid taught” group of students with the grades from groups of previous years in the same subject, by the same instructor, but totally in-person. We, as medical teachers, were excited about the possibility that the online teaching forced by the Covid-19 pandemic will develop in the system for delivering our lectures in the near future. Even considering that grades were not affected and pros such as comfort and participation, according to our study, students prefer in person education mainly because the direct contact and interaction with peers and teachers. info:eu-repo/semantics/publishedVersion

Polyphosphate (polyP) is a polymer of hundreds of phosphate residues present in all organisms. In mammals, polyP is involved in crucial physiological processes, including coagulation, inflammation, and stress response. However, after decades of research, the metabolic enzymes are still unknown. Here, we purify and identify Nudt3, a NUDIX family member, as the enzyme responsible for polyP phosphatase activity in mammalian cells. We show that Nudt3 shifts its substrate specificity depending on the cation; specifically, Nudt3 is active on polyP when Zn2+ is present. Nudt3 has in vivo polyP phosphatase activity in human cells, and importantly, we show that cells with altered polyP levels by modifying Nudt3 protein amount present reduced viability upon oxidative stress and increased DNA damage, suggesting that polyP and Nudt3 play a role in oxidative stress protection. Finally, we show that Nudt3 is involved in the early stages of embryo development in zebrafish. This work was supported by and is part of the I+D+i grant ref. PGC2018-096597-B-I00 (to J.J.) by the Spanish Ministerio de Ciencia e Innovación (MCIN). B.S.-M. was the recipient of a grant from the Agència de Gestió d’Ajuts Universitaris i de Recerca AGAUR ref. 2016FI_B 00025. H.J.J. was supported by the Deutsche Forschungsgemeinschaft (DFG) under Germany’s Excellence Strategy (CIBBS, EXC-2189, Project ID 390939984).

Purpose Cancer stem cells represent a cancer cell subpopulation that has been found to be associated with metastasis and chemoresistance. Therefore, it is vital to identify mechanisms regulating cancer stemness. Previously, we have shown that the atypical cyclin P (CCNP), also known as CNTD2, is upregulated in lung and colorectal cancers and is associated with a worse clinical prognosis. Given that other cyclins have been implicated in pluripotency regulation, we hypothesized that CCNP may also play a role in cancer stemness. Methods Cell line-derived spheroids, ex vivo intestinal organoid cultures and induced-pluripotent stem cells (iPSCs) were used to investigate the role of CCNP in stemness. The effects of CCNP on cancer cell stemness and the expression of pluripotency markers and ATP-binding cassette (ABC) transporters were evaluated using Western blotting and RT-qPCR assays. Cell viability was assessed using a MTT assay. The effects of CCNP on WNT targets were monitored by RNA-seq analysis. Data from publicly available web-based resources were also analyzed. Results We found that CCNP increases spheroid formation in breast, lung and colorectal cancers, and upregulates the expression of stemness (CD44, CD133) and pluripotency (SOX2, OCT4, NANOG) markers. In addition, we found that CCNP promotes resistance to anticancer drugs and induces the expression of multidrug resistance ABC transporters. Our RNA-seq data indicate that CCNP activates the WNT pathway, and that inhibition of this pathway abrogates the increase in spheroid formation promoted by CCNP. Finally, we found that CCNP knockout decreases OCT4 expression in iPSCs, further supporting the notion that CCNP is involved in stemness regulation. Conclusion Our results reveal CCNP as a novel player in stemness and as a potential therapeutic target in cancer.

There are two major pathways for repairing DNA double-strand breaks (DSBs): homologous directed recombination (HDR) and non-homologous end-joining (NHEJ). While NHEJ functions throughout the cell cycle, HDR is only possible during S/G2 phases, suggesting that there are cell cycle-specific mechanisms regulating the balance between the two repair systems. The regulation exerted by CDKs on HDR has been extensively demonstrated, and here we present evidence that the CDK Pho85, in association with the G1 cyclin Pcl1, phosphorylates Yku80 on Ser 623 to regulate NHEJ activity. Cells bearing a non-phosphorylatable version of Yku80 show increased NHEJ and reduced HDR activity. Accordingly, yku80S623A cells present diminished viability upon treatment with the DSB-producer bleomycin, specifically in the G2 phase of the cell cycle. Interestingly, the mutation of the equivalent residue in human Ku80 increases sensitivity to bleomycin in several cancer cell lines, suggesting that this mechanism is conserved in humans. Altogether, our results reveal a new mechanism whereby G1-CDKs mediate the choice between HDR and NHEJ repair pathways, putting the error prone NHEJ on a leash and enabling error free HDR in G2 when homologous sequences are available.

Cells require extra amounts of dNTPs to repair DNA after damage. Polyphosphate (polyP) is an evolutionary conserved linear polymer of up to several hundred inorganic phosphate (Pi) residues that is involved in many functions, including Pi storage. In the present article, we report on findings demonstrating that polyP functions as a source of Pi when required to sustain the dNTP increment essential for DNA repair after damage. We show that mutant yeast cells without polyP produce less dNTPs upon DNA damage and that their survival is compromised. In contrast, when polyP levels are ectopically increased, yeast cells become more resistant to DNA damage. More importantly, we show that when polyP is reduced in HEK293 mammalian cell line cells and in human dermal primary fibroblasts (HDFa), these cells become more sensitive to DNA damage, suggesting that the protective role of polyP against DNA damage is evolutionary conserved. In conclusion, we present polyP as a molecule involved in resistance to DNA damage and suggest that polyP may be a putative target for new approaches in cancer treatment or prevention. We would like to thank all the members of our group (E. Bállega, O. Mirallas, M. Ribeiro, A. Sánchez, B. Semper, and R. Carballar) for day-to-day talks, and Marta Pérez for technical assistance. The yeast ppn1Δ, ppn2Δ, ppx1Δ strain is a kind gift from A Mayer (Université de Lausanne). This work was supported by funding from the Spanish Government, with a MINECO grant (Ref: BFU 2013-44189-P) awarded to J. Clotet and a MICINN grant (Ref: BFU 2013-42895-P) awarded to E. Garí. J.M.M. was the recipient of a post-graduate Junior Faculty Fellowship from the UIC and l’Obra Social la Caixa.

Polyphosphate (polyP) is an abundant and physiologically important biomolecule for virtually any living cell. Therefore, determination of changes in cellular content of polyP is crucial for its functional characterization. Determination of cellular polyP has been performed by many different methods, and the lack of a standardized procedure is possibly responsible for the large dispersion of results found in the relevant literature. For a relatively simple organism, such as the yeast Saccharomyces cerevisiae, this variation can be up to 12-fold. polyP extraction and determination of free phosphate released by enzymatic degradation of the polymer is a method quite common and relatively straightforward for polyP determination. By using the yeast S. cerevisiae as model, we have experimentally evaluated the different steps in this procedure in order to identify critical issues that might explain the disparate reported results. As the main output of this evaluation we propose a straightforward and robust procedure that can be used as gold standard protocol for cellular polyP purification and determination from unicellular organisms, thus providing consistency to measurements and facilitating inter-laboratory comparisons and biological interpretation of the results.

Regulation of cell division is orchestrated by cyclins, which bind and activate their catalytic workmates, the cyclin-dependent kinases (CDKs). Cyclins have been traditionally defined by an oscillating (cyclic) pattern of expression and by the presence of a characteristic “cyclin box” that determines binding to the CDKs. Noteworthy, the Human Genome Sequence Project unveiled the existence of several other proteins containing the “cyclin box” domain. These potential “cyclins” have been named new, orphan or atypical, creating a conundrum in cyclins nomenclature. Moreover, although many years have passed after their discovery, the scarcity of information regarding these possible members of the family has hampered the establishment of criteria for systematization. Here, we discuss the criteria that define cyclins and we propose a classification and nomenclature update based on structural features, interactors, and phylogenetic information. The application of these criteria allows to systematically define, for the first time, the subfamily of atypical cyclins and enables the use of a common nomenclature for this extended family.

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that forms an active complex with cyclin Y (CCNY). Although both proteins have been recently implicated in cancer pathogenesis, it is still unclear how the CDK16/CCNY complex exerts its biological activity. To understand the CDK16/CCNY network, we used complementary proteomic approaches to identify potential substrates of this complex. We identified several candidates implicating the CDK16/CCNY complex in cytoskeletal dynamics, and we focused on the microtubule-associated protein regulator of cytokinesis (PRC1), an essential protein for cell division that organizes antiparallel microtubules and whose deregulation may drive genomic instability in cancer. Using analog-sensitive (AS) CDK16 generated by CRISPR-Cas9 mutagenesis in 293T cells, we found that specific inhibition of CDK16 induces PRC1 dephosphorylation at Thr481 and delocalization to the nucleus during interphase. The observation that CDK16 inhibition and PRC1 downregulation exhibit epistatic effects on cell viability confirms that these proteins can act through a single pathway. In conclusion, we identified PRC1 as the first substrate of the CDK16/CCNY complex and demonstrated that the proliferative function of CDK16 is mediated by PRC1 phosphorylation. As CDK16 is emerging as a critical node in cancer, our study reveals novel potential therapeutic targets., Cancer: Proteins that work together could reveal drug targets Studying the activity of proteins that work together to control cell division is revealing several that might be suitable targets for new drugs to fight cancer. Researchers led by Josep Clotet and Mariana Ribeiro at the International University of Catalonia, Barcelona, Spain, investigated the activities of the complex formed between two proteins, CDK16 and CCNY. CDK16 is an enzyme that modifies other molecules by adding phosphate groups (PO4) to them. CCNY is a protein that controls the activity of CDK16 and other proteins. Previous research has suggested a role for the complex in the development of cancer, but the mechanism has been unclear. The researchers found that the CDK16/CCNY complex activates proteins that control the network of microtubules in cells known as the cytoskeleton. One of these proteins, PRC1, is essential for cell division.

Polyphosphate (polyP) is a linear chain of up to hundreds of inorganic phosphate residues that is necessary for many physiological functions in all living organisms. In some bacteria, polyP supplies material to molecules such as DNA, thus playing an important role in biosynthetic processes in prokaryotes. In the present study, we set out to gain further insight into the role of polyP in eukaryotic cells. We observed that polyP amounts are cyclically regulated in Saccharomyces cerevisiae, and those mutants that cannot synthesise (vtc4Δ) or hydrolyse polyP (ppn1Δ, ppx1Δ) present impaired cell cycle progression. Further analysis revealed that polyP mutants show delayed nucleotide production and increased genomic instability. Based on these findings, we concluded that polyP not only maintains intracellular phosphate concentrations in response to fluctuations in extracellular phosphate levels, but also muffles internal cyclic phosphate fluctuations, such as those produced by the sudden demand of phosphate to synthetize deoxynucleotides just before and during DNA duplication. We propose that the presence of polyP in eukaryotic cells is required for the timely and accurate duplication of DNA.

Colorectal cancer (CRC) is one of the most common cancers worldwide, with 8–10% of these tumours presenting a BRAF (V600E) mutation. Cyclins are known oncogenes deregulated in many cancers, but the role of the new subfamily of atypical cyclins remains elusive. Here we have performed a systematic analysis of the protein expression levels of eight atypical cyclins in human CRC tumours and several cell lines, and found that CNTD2 is significantly upregulated in CRC tissue compared to the adjacent normal one. CNTD2 overexpression in CRC cell lines increases their proliferation capacity and migration, as well as spheroid formation capacity and anchorage-independent growth. Moreover, CNTD2 increases tumour growth in vivo on xenograft models of CRC with wild-type BRAF. Accordingly, CNTD2 downregulation significantly diminished the proliferation of wild-type BRAF CRC cells, suggesting that CNTD2 may represent a new prognostic factor and a promising drug target in the management of CRC.

Yeast cells have developed complex mechanisms to cope with extracellular insults. An increase in external osmolarity leads to activation of the stress-activated protein kinase Hog1, which is the main regulator of adaptive responses, such as gene expression and cell cycle progression, that are essential for cellular survival. Upon osmostress, the G1-to-S transition is regulated by Hog1 through stabilization of the cyclin-dependent kinase inhibitor Sic1 and the downregulation of G1 cyclin expression by an unclear mechanism. Here, we show that Hog1 interacts with and phosphorylates components of the core cell cycle transcriptional machinery such as Whi5 and the coregulator Msa1. Phosphorylation of these two transcriptional regulators by Hog1 is essential for inhibition of G1 cyclin expression, for control of cell morphogenesis, and for maximal cell survival upon stress. The control of both Whi5 and Msa1 by Hog1 also revealed the necessity for proper coordination of budding and DNA replication. Thus, Hog1 regulates G1 cyclin transcription upon osmostress to ensure coherent passage through Start.

The molecular chaperones Hsp70 and Hsp90 participate in many important cellular processes, including how cells respond to DNA damage. Here we show the results of applied quantitative affinity-purification mass spectrometry (AP-MS) proteomics to understand the protein network through which Hsp70 and Hsp90 exert their effects on the DNA damage response (DDR). We characterized the interactomes of the yeast Hsp70 isoform Ssa1 and Hsp90 isoform Hsp82 before and after exposure to methyl methanesulfonate. We identified 256 chaperone interactors, 146 of which are novel. Although the majority of chaperone interaction remained constant under DNA damage, 5 proteins (Coq5, Ast1, Cys3, Ydr210c and Rnr4) increased in interaction with Ssa1 and/or Hsp82. This data presented here are related to [1] (Truman et al., in press). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (Vizcaino et al. (2013) [2]) with the dataset identifier PXD001284. info:eu-repo/semantics/acceptedVersion

The highly conserved molecular chaperones Hsp90 and Hsp70 are indispensible for folding and maturation of a significant fraction of the proteome, including many proteins involved in signal transduction and stress response. To examine the dynamics of chaperone–client interactions after DNA damage, we applied quantitative affinity-purification mass spectrometry (AP-MS) proteomics to characterize interactomes of the yeast Hsp70 isoform Ssa1 and Hsp90 isoform Hsp82 before and after exposure to methyl methanesulfonate. Of 256 proteins identified and quantified via 16O/18O labeling and LC–MS/MS, 142 are novel Hsp70/90 interactors. Nearly all interactions remained unchanged or decreased after DNA damage, but 5 proteins increased interactions with Ssa1 and/or Hsp82, including the ribonucleotide reductase (RNR) subunit Rnr4. Inhibiting Hsp70 or 90 chaperone activity destabilized Rnr4 in yeast and its vertebrate homolog hRMM2 in breast cancer cells. In turn, pre-treatment of cancer cells with chaperone inhibitors sensitized cells to the RNR inhibitor gemcitabine, suggesting a novel chemotherapy strategy. All MS data have been deposited in the ProteomeXchange with identifier PXD001284. Biological significance This study provides the dynamic interactome of the yeast Hsp70 and Hsp90 under DNA damage which suggest key roles for the chaperones in a variety of signaling cascades. Importantly, the cancer drug target ribonucleotide reductase was shown to be a client of Hsp70 and Hsp90 in both yeast and breast cancer cells. As such, this study highlights the potential of a novel cancer therapeutic strategy that exploits the synergy of chaperone and ribonucleotide reductase inhibitors.

Deciphering the molecular mechanisms that connect cell cycle progression and nucleocytoplasmic transport is of particular interest: this intertwined relationship, once understood, may provide useful insight on the diseases resulting from the malfunction of these processes. In the present study we report on findings that indicate a biochemical connection between the cell cycle regulator CDK Pho85 and Ran-GTPase Gsp1, an essential nucleocytoplasmic transport component. When Gsp1 cannot be phosphorylated by Pho85, the cell cycle progression is impaired. Accordingly, a nonphosphorylatable version of Gsp1 abnormally localizes to the nucleus, which impairs the nuclear transport of molecules, including key components of cell cycle progression. Furthermore, our results suggest that the physical interaction of Gsp1 and the Kap95 karyopherin, essential to the release of nuclear cargoes, is altered. Altogether, the present findings point to the involvement of a biochemical mechanism in the interlocked regulation of the cell cycle and nuclear transport.

Our understanding of lifespan has benefited enormously from the study of a simple model, the yeast Saccharomyces cerevisiae. Although a unicellular organism, yeasts undergo many of the processes directly related with aging that to some extent are conserved in mammalian cells. Nutrient-limiting conditions have been involved in lifespan extension, especially in the case of caloric restriction, which also has a direct impact on cell cycle progression. In fact, other environmental stresses (osmotic, oxidative) that interfere with normal cell cycle progression also influence the lifespan of cells, indicating a relationship between lifespan and cell cycle control. In the present review we compile and discuss new findings related to how cell cycle progression is regulated by other nutrients. We centred this review on the analysis of phosphate, also give some attention to nitrogen, and the impact of these nutrients on lifespan.

As lung cancer has increased to the most common cause of cancer death worldwide, prognostic biomarkers and effective targeted treatments remain lacking despite advances based on patients’ stratification. Multiple core cyclins, best known as drivers of cell proliferation, are commonly deregulated in lung cancer where they may serve as oncogenes. The recent expansion of the cyclin family raises the question whether new members might play oncogenic roles as well. Here, we investigated the protein levels of eight atypical cyclins in lung cancer cell lines and formalin-fixed and paraffin-embedded (FFPE) human tumors, as well as their functional role in lung cancer cells. Of the new cyclins evaluated, CNTD2 was significantly overexpressed in lung cancer compared to adjacent normal tissue, and exhibited a predominant nuclear location. CNTD2 overexpression increased lung cancer cell viability, Ki-67 intensity and clonogenicity and promoted lung cancer cell migration. Accordingly, CNTD2 enhanced tumor growth in vivo on A549 xenograft models. Finally, the analysis of gene expression data revealed a high correlation between elevated levels of CNTD2 and decreased overall survival in lung cancer patients. Our results reveal CNTD2 as a new oncogenic driver in lung cancer, suggesting value as a prognostic biomarker and therapeutic target in this disease.

Progression through the G1 phase of the cell cycle is controlled by diverse cyclin-dependent kinases (CDKs) that might be associated to numerous cyclin isoforms. Given such complexity, regulation of cyclin degradation should be crucial for coordinating progression through the cell cycle. In Saccharomyces cerevisiae, SCF is the only E3 ligase known to date to be involved in G1 cyclin degradation. Here, we report the design of a genetic screening that uncovered Dma1 as another E3 ligase that targets G1 cyclins in yeast. We show that the cyclin Pcl1 is ubiquitinated in vitro and in vivo by Dma1, and accordingly, is stabilized in dma1 mutants. We demonstrate that Pcl1 must be phosphorylated by its own CDK to efficiently interact with Dma1 and undergo degradation. A nonphosphorylatable version of Pcl1 accumulates throughout the cell cycle, demonstrating the physiological relevance of the proposed mechanism. Finally, we present evidence that the levels of Pcl1 and Cln2 are independently controlled in response to nutrient availability. This new previously unknown mechanism for G1 cyclin degradation that we report here could help elucidate the specific roles of the redundant CDK-cyclin complexes in G1., This work was supported by Spanish Ministry of Science and Innovation Grant BFU 2009-09728, now known as the Ministry of Economy and Competitiveness.

Phosphorus is a pivotal element in all biochemical systems: it serves to store metabolic energy as ATP, it forms the backbone of genetic material such as RNA and DNA, and it separates cells from the environment as phospholipids. In addition to this "big hits", phosphorus has recently been shown to play an important role in other important processes such as cell cycle regulation. In the present review, we briefly summarize the biological processes in which phosphorus is involved in the yeast Saccharomyces cerevisiae before discussing our latest findings on the role of this element in the regulation of DNA replication in this eukaryotic model organism. We describe both the role of phosphorus in the regulation of G1 progression by means of the Cyclin Dependent Kinase (CDK) Pho85 and the stabilization of the cyclin Cln3, as well as the role of other molecule composed of phosphorus-the polyphosphate-in cell cycle progression, dNTP synthesis, and genome stability. Given the eminent role played by phosphorus in life, we outline the future of phosphorus in the context of one of the main challenges in human health: cancer treatment. [Int Microbiol 19(3):133-141 (2016)].

Phosphate is one of the essential elements supporting life. Cells accumulate phosphate in the form of a molecule called polyphosphate (polyP), which carries many functions in the physiology of cells that have not been wholly elucidated. Polyphosphate is present in all the types of cells from bacteria to mammals. It consists of a linear polymer constructed with anywhere from a few to hundreds of inorganic phosphate (Pi) molecules linked by phosphoanhydride bonds. Although polyP was described many years ago, difficulties in the study of its roles, most likely due to the many processes polyP is involved in and incomplete information obtained from multiple models and organisms relegate polyP into oblivion. But now, several interesting pieces of evidence are resurrecting the polyP as a key molecule in processes, such as protein folding, carbon metabolism, cell cycle progression, dNTP synthesis, and genomic stability. In this contribution, in addition to briefly summarize the polyP history and roles, we discuss its involvement in supporting cell cycle progression and genomic stability as well as the implications for the truthful replication of genomes.

Resveratrol is a polyphenol suggested to play a protective role against ageing and age-related diseases. We demonstrate that administering low-doses of resveratrol causes ROS accumulation and transcriptional changes in yeast cells and human adipocytes. These changes in gene expression depend on the oxidative transcription factor Yap1p. In particular, resveratrol induces expression of Yap1p gene targets, such as TRX2, TRR1 or AHP1, in a Yap1p-dependent mode. Under resveratrol treatment, Yap1p is phosphorylated and accumulated in the nucleus. Yap1p knockout causes resveratrol sensitivity, which totally depends on the presence of the C-terminal region of Yap1p. Thus, resveratrol may enhance cellular lifespan by hormetic ROS accumulation, which leads to strengthening the cells' antioxidant capacity.

Polyphosphate (polyP) is a linear chain of up to hundreds of inorganic phosphate residues that is necessary for many physiological functions in all living organisms. In some bacteria, polyP supplies material to molecules such as DNA, thus playing an important role in biosynthetic processes in prokaryotes. In the present study, we set out to gain further insight into the role of polyP in eukaryotic cells. We observed that polyP amounts are cyclically regulated in Saccharomyces cerevisiae, and those mutants that cannot synthesise (vtc4Δ) or hydrolyse polyP (ppn1Δ, ppx1Δ) present impaired cell cycle progression. Further analysis revealed that polyP mutants show delayed nucleotide production and increased genomic instability. Based on these findings, we concluded that polyP not only maintains intracellular phosphate concentrations in response to fluctuations in extracellular phosphate levels, but also muffles internal cyclic phosphate fluctuations, such as those produced by the sudden demand of phosphate to synthetize deoxynucleotides just before and during DNA duplication. We propose that the presence of polyP in eukaryotic cells is required for the timely and accurate duplication of DNA.

In order to study stress–strain tensors, we consider their representations as pairs of symmetric 3 × 3-matrices and the space of such pairs of matrices partitioned into equivalence classes corresponding to change of bases. We see that these equivalence classes are differentiable submanifolds; in fact, orbits under the action of a Lie group. We compute their dimension and obtain miniversal deformations. Finally, we prove that the space of coaxial stress–strain tensors is a finite union of differentiable submanifolds. Copyright © 2008 John Wiley & Sons, Ltd.

We consider the set of quadruples of matrices defining regular singular linear time-invariant dynamical systems under restricted system equivalence and derive miniversal deformations from a basis of the normal space to orbits under the Lie group action related to this equivalence relation. A lower bound and an upper bound for the dimension of the orbits are obtained. We conclude with examples and further comments about genericity.

3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase adopts a (betaalpha)(8) TIM barrel structure with an additional beta9, alpha11 and alpha12 helices. Location of HMG part of the substrate has been suggested but the binding mode for the CoA moiety remains to be resolved. As mutation F305 fs(-2), which involves the last 21 residues of the protein, and mutation K48N caused 3-hydroxy-3-methylglutaric aciduria in two patients, we examined the role of the C-terminal end and Lys(48) in enzyme activity. Expression studies of various C-terminal-end-deleted and K48N-mutated proteins revealed that residues 311-313 (localized in the loop between alpha11 and alpha12 helices) and Lys(48) are essential for enzyme activity. An in silico docking model locating HMG-CoA on the surface of the enzyme implicates Asn(311) and Lys(313) in substrate binding by establishing multiple polar contacts with phosphate and ribose groups of adenosine, and Lys(48) by contacting the carboxyl group of the panthotenic acid moiety.

No necessary and sufficient condition for reachability of switched linear singular systems has been found, exceptuating the case of the so-called “equisingular systems”. Such a condition is not valid in the general case, as examples show.

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to osmostress leads to activation of the Hog1 SAPK, which controls cell cycle at G1 by the targeting of Sic1. Here, we show that survival to osmostress also requires regulation of G2 progression. Activated Hog1 interacts and directly phosphorylates a residue within the Hsl7-docking site of the Hsl1 checkpoint kinase, which results in delocalization of Hsl7 from the septin ring and leads to Swe1 accumulation. Upon Hog1 activation, cells containing a nonphosphorylatable Hsl1 by Hog1 are unable to promote Hsl7 delocalization, fail to arrest at G2 and become sensitive to osmostress. Together, we present a novel mechanism that regulates the Hsl1-Hsl7 complex to integrate stress signals to mediate cell cycle arrest and, demonstrate that a single MAPK coordinately modulates different cell cycle checkpoints to improve cell survival upon stress.

Inactivation of HAL3 in the absence of SIT4 function leads to cell cycle arrest at the G1–S transition. To identify genes potentially involved in the control of this phase of the cell cycle, a screening for multicopy suppressors of a conditional sit4 hal3 mutant (strain JC002) has been developed. The screening yielded several genes known to perform key roles in cell cycle events, such as CLN3, BCK2 or SWI4, thus proving its usefulness as a tool for this type of studies. In addition, this approach allowed the identification of additional genes, most of them not previously related to the regulation of G1–S transition or even without known function (named here as VHS1-3, for viable in a hal3sit4 background). Several of these gene products are involved in phospho-dephosphorylation processes, including members of the protein phosphatase 2A and protein phosphatases 2C families, as well as components of the Hal5 protein kinase family. The ability of different genes to suppress sit4 phenotypes (such as temperature sensitivity and growth on non-fermentable carbon sources) or to mimic the functions of Hal3 was evaluated. The possible relationship between the known functions of these suppressor genes and the progress through the G1–S transition is discussed. Copyright © 2002 John Wiley & Sons, Ltd.

The aim of this paper is to study what happens when a slight perturbation affects the coefficients of a quadratic equation defining a variety (a quadric) in R n . Structurally stable quadrics are those such that a small perturbation on the coefficients of the equation defining them does not give rise to a “different” (in some sense) set of points. In particular, we characterize structurally stable quadrics and give the “bifurcation diagrams” of the non-stable ones (showing which quadrics meet all of their neighbourhoods), when dealing with the “affine” and “metric” equivalence relations. This study can be applied to the case where a set of points, which constitute the set of solutions of a problem, is defined by a quadratic equation whose coefficients are given with parameter uncertainty.

Carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) I, which facilitate the transport of medium- and long-chain fatty acids through the peroxisomal and mitochondrial membranes, are physiologically inhibited by malonyl-CoA. Using an “in silico” macromolecular docking approach, we built a model in which malonyl-CoA could be attached near the catalytic core. This disrupts the positioning of the acyl-CoA substrate in the channel in the model reported for both proteins (Morillas, M., Gomez-Puertas, P., Roca, R., Serra, D., Asins, G., Valencia, A., and Hegardt, F. G. (2001) J. Biol. Chem. 276, 45001–45008). The putative malonyl-CoA domain contained His340, implicated together with His131 in COT malonyl-CoA sensitivity (Morillas, M., Clotet, J., Rubi, B., Serra, D., Asins, G., Arino, J., and Hegardt F. G. (2000) FEBS Lett. 466, 183–186). When we mutated COT His131 the IC50increased, and malonyl-CoA competed with the substrate decanoyl-CoA. Mutation of COT Ala332, present in the domain 8 amino acids away from His340, decreased the malonyl-CoA sensitivity of COT. The homologous histidine and alanine residues of L-CPT I, His277, His483, and Ala478 were also mutated, which decreased malonyl-CoA sensitivity. Natural mutation of Pro479, which is also located in the malonyl-CoA predicted site, to Leu in a patient with human L-CPT I hereditary deficiency, modified malonyl-CoA sensitivity. We conclude that this malonyl-CoA domain is present in both COT and L-CPT I proteins and might be the site at which malonyl-CoA interacts with the substrate acyl-CoA. Other malonyl-CoA non-inhibitable members of the family, CPT II and carnitine acetyltransferase, do not contain this domain.

A screening for multicopy suppressors of the G(1)/S blockage of a conditional sit4 hal3 mutant yielded the NHA1 gene, encoding a Na(+),K(+)/H(+) antiporter, composed of a transmembrane domain and a large carboxyl-terminal tail, which has been related to cation detoxification processes. Expression of either the powerful Saccharomyces cerevisiae Ena1 Na(+)/H(+)-ATPase or the Schizosaccharomyces pombe Sod2 Na(+)/H(+) antiporter, although increasing tolerance to sodium, was unable to mimic the Nha1 function in the cell cycle. Mutation of the conserved Asp residues Asp(266)-Asp(267) selectively abolished Na(+) efflux without modifying K(+) efflux and did not affect the capacity of Nha1 to relieve the G(1) blockage. Mutagenesis analysis revealed that the region near the carboxyl-terminal end of Nha1 comprising residues 800-948 is dispensable for sodium detoxification but necessary for transport of K(+) cations. Therefore, this portion of the protein contains structural elements that selectively modulate Nha1 antiporter functions. This region is also required for Nha1 to function in the cell cycle. However, expression of the closely related Cnh1 antiporter from Candida albicans, which also contains a long carboxyl-terminal extension, although allowing efficient K(+) transport does not relieve cell cycle blockage. This indicates that although the determinants for Nha1-mediated regulation of potassium transport and the cell cycle map very closely in the protein, most probably the function of Nha1 on cell cycle is independent of its ability to extrude potassium cations.

The gene pzl-1 from the filamentous fungus Neurospora crassa encodes a putative Ser/Thr protein phosphatase that is reminiscent of the Ppz1/Ppz2 and Pzh1 phosphatases from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The entire PZL-1 protein, as well as its carboxyl-terminal domain, have been expressed in Escherichia coli as active protein phosphatases. To characterize its cellular role, PZL-1 was also expressed in Sz. pombe and in S. cerevisiae. Expression of PZL-1 in S. cerevisiae from the PPZ1 promoter was able to rescue the altered sensitivity to caffeine and lithium ions of a ppz1 strain. Furthermore, high copy number expression of PZL-1 alleviated the lytic phenotype of a S. cerevisiae slt2/mpk1 mitogen-activated protein (MAP) kinase mutant, similarly to that described for PPZ1, and mimicked the effects of high levels of Ppz1 on cell growth. Expression of PZL-1 in fission yeast from a weak version of the nmt1 promoter fully rescued the growth defect of a pzh1Δ strain in high potassium, but only partially complemented the sodium-hypertolerant phenotype. Strong overexpression of the N. crassa phosphatase in Sz. pombe affected cell growth and morphology. Therefore, PZL-1 appears to fulfil every known function carried out by its S. cerevisiae counterpart, despite the marked divergence in sequence within their NH2-terminal moieties. Copyright © 2000 John Wiley & Sons, Ltd.